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1.  Thermoanaerobacter thermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates 
Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using “omic”-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species “omic” comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.
PMCID: PMC3957603  PMID: 24362431
2.  Proteomics-based metabolic modeling and characterization of the cellulolytic bacterium Thermobifida fusca 
BMC Systems Biology  2014;8:86.
Thermobifida fusca is a cellulolytic bacterium with potential to be used as a platform organism for sustainable industrial production of biofuels, pharmaceutical ingredients and other bioprocesses due to its capability of potential to convert plant biomass to value-added chemicals. To best develop T. fusca as a bioprocess organism, it is important to understand its native cellular processes. In the current study, we characterize the metabolic network of T. fusca through reconstruction of a genome-scale metabolic model and proteomics data. The overall goal of this study was to use multiple metabolic models generated by different methods and comparison to experimental data to gain a high-confidence understanding of the T. fusca metabolic network.
We report the generation of three versions of a metabolic model of Thermobifida fusca sp. XY developed using three different approaches (automated, semi-automated, and proteomics-derived). The model closest to in vivo growth was the proteomics-derived model that consists of 975 reactions involving 1382 metabolites and account for 316 EC numbers (296 genes). The model was optimized for biomass production with the optimal flux of 0.48 doublings per hour when grown on cellobiose with a substrate uptake rate of 0.25 mmole/h. In vivo activity of the DXP pathway for terpenoid biosynthesis was also confirmed using real-time PCR.
iTfu296 provides a platform to understand and explore the metabolic capabilities of the actinomycete T. fusca for the potential use in bioprocess industries for the production of biofuel and pharmaceutical ingredients. By comparing different model reconstruction methods, the use of high-throughput proteomics data as a starting point proved to be the most accurate to in vivo growth.
PMCID: PMC4236713  PMID: 25115351
Metabolic Modeling; Flux Balance Analysis; Constraint Based Modeling; Actinomycete; Thermobifida fusca; Proteomics Profiling; Terpenoids Biosynthesis Pathway; DXP Pathway; Mevalonate Pathway; Biofuel
3.  Blood Pressure Trajectories in Early Adulthood and Subclinical Atherosclerosis in Middle Age 
Single measures of blood pressure (BP) levels are associated with the development of atherosclerosis; however, long-term patterns in BP and their impact on CVD risk are poorly characterized.
To identify common BP trajectories throughout early adulthood and to determine their association with presence of coronary artery calcification (CAC) during middle age.
We used data from the CARDIA study from baseline in 1985-1986 through 25 years of follow-up (2010-2011).
Prospective cohort study
CARDIA participants were Black and White men and women aged 18-30 years at baseline.
We examined systolic BP, diastolic BP, and mid-BP [calculated as (SBP+DBP)/2 and an important marker of CHD risk among younger populations] at baseline and years 2, 5, 7, 10, 15, 20, and 25. Latent mixture modeling was used to identify trajectories in SBP, DBP and mid-BP over time.
Main Outcome Measure
Coronary artery calcification greater than or equal to Agatston score of 100 Agatston units at year 25.
Among 4,681 participants, we identified 5 distinct mid-BP trajectories: Low-Stable (22% [95% CI 19.9-23.7], n=987), Moderate-Stable (42% [40.3-44.3], n=2,085), Moderate-Increasing (12% [10.4-14.0], n=489), Elevated-Stable (19% [17.1-20.0], n=903) and Elevated-Increasing (5% [4.0-5.5], n=217). As compared to the Low-Stable group, trajectories with elevated BP levels had greater odds of having CAC >100. Adjusted odds ratios (95% CI) were 1.44 (0.83-2.49) for Moderate-Stable, 1.86 (0.91-3.82) for Moderate-Increasing, 2.28 (1.24-3.70) for Elevated-Stable, and 3.70 (1.66-8.20) for Elevated-Increasing groups. The adjusted prevalence of CAC ≥ 100 was 5.8% in the Low-Stable group. These ORs represent an absolute increase of 2.7%, 5%, 6.3% and 12.9% for the prevalence of CAC ≥100 for the Moderate-Stable, Moderate-Increasing, Elevated Stable and Elevated Increasing groups respectively as compared to the Low-Stable Group. Associations were not altered after adjustment for baseline and year 25 BP. Findings were similar for trajectories of isolated systolic BP trajectories, but were attenuated for diastolic BP trajectories.
Conclusions and Relevance
BP trajectories throughout young adulthood vary and higher BP trajectories were associated with an increased risk of CAC in middle age. Long-term trajectories in BP may assist in more accurate identification of individuals with subclinical atherosclerosis.
PMCID: PMC4122296  PMID: 24496536
blood pressure; calcium; epidemiology; risk factors
4.  Enhanced whole genome sequence and annotation of Clostridium stercorarium DSM8532T using RNA-seq transcriptomics and high-throughput proteomics 
BMC Genomics  2014;15(1):567.
Growing interest in cellulolytic clostridia with potential for consolidated biofuels production is mitigated by low conversion of raw substrates to desired end products. Strategies to improve conversion are likely to benefit from emerging techniques to define molecular systems biology of these organisms. Clostridium stercorarium DSM8532T is an anaerobic thermophile with demonstrated high ethanol production on cellulose and hemicellulose. Although several lignocellulolytic enzymes in this organism have been well-characterized, details concerning carbohydrate transporters and central metabolism have not been described. Therefore, the goal of this study is to define an improved whole genome sequence (WGS) for this organism using in-depth molecular profiling by RNA-seq transcriptomics and tandem mass spectrometry-based proteomics.
A paired-end Roche/454 WGS assembly was closed through application of an in silico algorithm designed to resolve repetitive sequence regions, resulting in a circular replicon with one gap and a region of 2 kilobases with 10 ambiguous bases. RNA-seq transcriptomics resulted in nearly complete coverage of the genome, identifying errors in homopolymer length attributable to 454 sequencing. Peptide sequences resulting from high-throughput tandem mass spectrometry of trypsin-digested protein extracts were mapped to 1,755 annotated proteins (68% of all protein-coding regions). Proteogenomic analysis confirmed the quality of annotation and improvement pipelines, identifying a missing gene and an alternative reading frame. Peptide coverage of genes hypothetically involved in substrate hydrolysis, transport and utilization confirmed multiple pathways for glycolysis, pyruvate conversion and recycling of intermediates. No sequences homologous to transaldolase, a central enzyme in the pentose phosphate pathway, were observed by any method, despite demonstrated growth of this organism on xylose and xylan hemicellulose.
Complementary omics techniques confirm the quality of genome sequence assembly, annotation and error-reporting. Nearly complete genome coverage by RNA-seq likely indicates background DNA in RNA extracts, however these preps resulted in WGS enhancement and transcriptome profiling in a single Illumina run. No detection of transaldolase by any method despite xylose utilization by this organism indicates an alternative pathway for sedoheptulose-7-phosphate degradation. This report combines next-generation omics techniques to elucidate previously undefined features of substrate transport and central metabolism for this organism and its potential for consolidated biofuels production from lignocellulose.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-567) contains supplementary material, which is available to authorized users.
PMCID: PMC4102724  PMID: 24998381
Genome; Proteome; Transcriptome; RNA-seq; Tandem mass spectrometry; Proteogenomics; Glycolysis; Pentose phosphate pathway; Transaldolase
5.  Associations Between a Parental History of Premature Cardiovascular Disease and Coronary Calcium and Carotid Intima-Media Thickness: The Coronary Artery Risk Development In Young Adults (CARDIA) Study 
It is unclear if associations between a parental history of premature CVD (pCVD) and subclinical atherosclerosis are attenuated by adjustment for long-term risk factors levels through middle adulthood.
Prospective community-based cohort study
CARDIA participants who attended the year 20 exam (N=2283, mean age 45 years) were grouped by pCVD status: maternal only, paternal only, any parental, and no parental history (referent). We used separate logistic regression models, adjusted for average risk factor levels over 20 years' follow-up to assess associations of parental pCVD and subclinical atherosclerosis in offspring.
White participants with any parental history of pCVD had a higher odds of CAC>0 than participants with no parental history (OR 1.55; 95% CI, 1.01-2.37). This was largely driven by the association of a paternal history of pCVD with CAC>0 (OR 2.15; 95% CI, 1.42-3.23), which was minimally attenuated by multivariable adjustment (OR 2.09; 95% CI, 1.31-3.32). Similarly, adjusted associations between parental pCVD and IMT > 90%tile were observed in white participants with a paternal history of pCVD (OR=1.93; 95% CI, 1.10-3.39) and any parental history pCVD (OR 1.67; 95% CI, 1.02-2.74). No significant associations between a parental history of pCVD and the odds of subclinical atherosclerosis were observed in black participants.
Parental pCVD is independently associated with early development of subclinical atherosclerosis; these associations may be race-specific for participants in their 5th decade of life.
PMCID: PMC3779512  PMID: 23027592
Family History of Premature Cardiovascular Disease; Coronary Artery Calcium; Carotid Intima-Media Thickness
6.  Coronary Heart Disease Risks Associated with High Levels of HDL Cholesterol 
The association between high‐density lipoprotein cholesterol (HDL‐C) and coronary heart disease (CHD) events is not well described in individuals with very high levels of HDL‐C (>80 mg/dL).
Methods and Results
Using pooled data from 6 community‐based cohorts we examined CHD and total mortality risks across a broad range of HDL‐C, including values in excess of 80 mg/dL. We used Cox proportional hazards models with penalized splines to assess multivariable, adjusted, sex‐stratified associations of HDL‐C with the hazard for CHD events and total mortality, using HDL‐C 45 mg/dL and 55 mg/dL as the referent in men and women, respectively. Analyses included 11 515 men and 12 925 women yielding 307 245 person‐years of follow‐up. In men, the association between HDL‐C and CHD events was inverse and linear across most HDL‐C values; however at HDL‐C values >90 mg/dL there was a plateau effect in the pattern of association. In women, the association between HDL‐C and CHD events was inverse and linear across lower values of HDL‐C, however at HDL‐C values >75 mg/dL there were no further reductions in the hazard ratio point estimates for CHD. In unadjusted models there were increased total mortality risks in men with very high HDL‐C, however mortality risks observed in participants with very high HDL‐C were attenuated after adjustment for traditional risk factors.
We did not observe further reductions in CHD risk with HDL‐C values higher than 90 mg/dL in men and 75 mg/dL in women.
PMCID: PMC4187512  PMID: 24627418
CHD events; total mortality; very‐high HDL‐C
7.  Proteomic characterization of serine hydrolase activity and composition in normal urine 
Clinical Proteomics  2013;10(1):17.
Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.
Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.
Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.
This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.
PMCID: PMC4225696  PMID: 24237849
Activity-based protein profiling; Catabolomics; Fluorophosphonate probe; Mass spectrometry
8.  High Density GWAS for LDL Cholesterol in African Americans Using Electronic Medical Records Reveals a Strong Protective Variant in APOE 
Only one LDL-C GWAS has been reported in African Americans. We performed a GWAS of LDL-C in African Americans using data extracted from electronic medical records (EMR) in the eMERGE network. African Americans were genotyped on the Illumina 1M chip. All LDL-C measurements, prescriptions, and diagnoses of concomitant disease were extracted from EMR. We created two analytic datasets; one dataset having median LDL-C calculated after the exclusion of some lab values based on co-morbidities and medication (n = 618) and another dataset having median LDL-C calculated without any exclusions (n = 1249). Rs7412 in APOE was strongly associated with LDL-C at levels of GWAS significance in both datasets (p < 5 X 10−8). In the dataset with exclusions, a decrease of 20.0 mg/dl per minor allele was observed. The effect size was attenuated (12.3 mg/dl) in the dataset without any lab values excluded. Although other signals in APOE have been detected in previous GWAS, this large and important SNP association has not been well detected in large GWAS because rs7412 was not included on many genotyping arrays. Use of median LDL-C extracted from EMR after exclusions for medications and co-morbidities increased the percentage of trait variance explained by genetic variation.
PMCID: PMC3521536  PMID: 23067351
GWAS; LDL; electronic medical records
9.  Associations of Non-Invasive Measures of Arterial Compliance and Ankle-Brachial Index: The Multi-Ethnic Study of Atherosclerosis (MESA) 
American journal of hypertension  2012;25(5):535-541.
The association between measures of arterial compliance and peripheral arterial disease (PAD) is unclear. Early changes in arterial wall compliance could be a useful marker of patients at high risk for developing lower extremity atherosclerosis.
We used linear and logistic regression models on baseline data from 2803 female and 2558 male participants in the Multi-Ethnic Study of Atherosclerosis (MESA) to study associations between tonometry-derived baseline measures of arterial compliance (large artery compliance [C1] and small artery compliance [C2]) and the baseline ankle-brachial index (ABI), as well as change in the ABI over approximately 3 years of follow up.
In cross-sectional analyses, lower C1 and C2 values, indicating poorer arterial compliance, were associated with lower ABI. There were significant linear trends across strata of ABI, especially in C2 which ranged from 3.7ml/mmHg × 100 (95% confidence interval (CI) 3.3 to 4.2) in women with an ABI < 0.90 to 4.2ml/mmHg × 100 (95% CI 4.1 to 4.3 p<0.001) in women with ABI 1.10 - <1.40. Similar significant trends (p<0.001) were seen in men. In prospective analyses, those with the lowest tertile of C2 values at baseline had a greater multivariable-adjusted odds for decline in ABI of ≥ 0.15 over 3 years compared to those with the highest C2 values at baseline (OR 1.80 95% CI 1.23–2.64).
We observed that less compliant arteries were significantly associated with low ABI in cross-sectional analysis and with greater decline in ABI over time.
PMCID: PMC3748962  PMID: 22357412
Ankle-Brachial Index; Arterial Compliance; Peripheral Arterial Disease
10.  Lifetime Risk and Years Lived Free of Total Cardiovascular Disease 
Estimates of lifetime risk (LTR) for total cardiovascular disease (tCVD) may provide projections of the future population burden of cardiovascular disease and may assist in clinician-patient risk communication. To date, no LTR estimates of tCVD have been reported.
To calculate LTR estimates of tCVD by index age [45, 55, 65, 75 years(y)] and risk factor strata and to estimate years lived free of CVD across risk factor strata.
Design, Setting, and Participants
Pooled survival analysis of up to 905,115 person-years of data from 1964 through 2008 from 5 NHLBI-funded community-based cohorts: Framingham Heart Study, Framingham Offspring Study, Atherosclerosis Risk in Communities Study, Chicago Heart Association Detection Project in Industry Study and Cardiovascular Health Study.
All participants free of CVD at baseline with risk factor data (blood pressure (BP), total cholesterol (TC), diabetes and smoking status) and tCVD outcome data
Outcome Measures
Any tCVD event (including fatal and non-fatal coronary heart disease, all forms of stroke, congestive heart failure and other CVD deaths)
At an index age of 45y, overall LTR for tCVD was 60.3% (95% CI, 59.3 to 61.2) for men and 55.6% (95% CI, 54.5 to 56.7) for women. Men had higher LTR estimates than women across all index ages. At index ages 55 and 65y, men and women with ≥1 elevated risk factor (BP 140-149/90-99 mmHg or TC 200-239 mg/dL but no diabetes or smoking), or 1, or ≥ 2 major risk factors (BP ≥ 160/100mmHg or on treatment; TC ≥ 240mg/dL or on treatment, diabetes mellitus, or current smoking) had LTR estimates to age 95y that exceeded 50%. Despite an optimal risk factor profile (BP < 120/80 mmHg, TC < 180 mg/dL, and no smoking or diabetes) men and women at an index age of 55y had LTR for total CVD to age 85y > 40% and 30% respectively. Compared with participants with ≥ 2 major risk factors, those with an optimal risk factor profile lived up to 14y longer free of tCVD.
LTR estimates for tCVD are high (>30%) for all individuals, even those with optimal risk factors in middle age. However, maintenance of optimal risk factor levels in middle age is associated with substantially longer morbidity-free survival.
PMCID: PMC3748966  PMID: 23117780
Lifetime Risk; Cardiovascular Disease; Compression of Morbidity
11.  Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation 
Journal of Virology  2013;87(2):1049-1060.
Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.
PMCID: PMC3554053  PMID: 23135712
12.  Quantification of the Host Response Proteome after Mammalian Reovirus T1L Infection 
PLoS ONE  2012;7(12):e51939.
All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular “transcriptome.” We used SILAC (stable isotope labeling by amino acids in cell culture), combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L). 3,076 host proteins were detected at 6hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.
PMCID: PMC3519901  PMID: 23240068
13.  Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression 
BMC Microbiology  2012;12:214.
Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.
Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.
Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.
PMCID: PMC3492117  PMID: 22994686
14.  Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes 
BMC Immunology  2012;13:21.
Lymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration.
Microscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of β1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the β2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1.
The present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated adhesion.
PMCID: PMC3447661  PMID: 22510515
T lymphocytes; Chemotaxis; CXCL12; Pseudopodia; Glia maturation factor gamma; GMFG; Proteomics; ShRNAmir; Adhesion
15.  Granzyme B Inhibits Vaccinia Virus Production through Proteolytic Cleavage of Eukaryotic Initiation Factor 4 Gamma 3 
PLoS Pathogens  2011;7(12):e1002447.
Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD1408S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.
Author Summary
Lymphocytes, a type of white blood cell, are the major killer of virus-infected cells. Lymphocytes secrete proteins like granzyme B that are responsible for the destruction of the virus-infected host cell. However, killing an infected cell through this pathway may take several hours, thus allowing viral replication to occur while the cell is in the process of dying. In this study, we identified a new role of granzyme B in preventing viral replication during the killing process. We found that granzyme B disables the ability of the host cell to make new proteins, including viral proteins of infected cells. Thus, granzyme B is able to halt the production of new viruses by inhibiting protein production.
PMCID: PMC3240606  PMID: 22194691
16.  Requirement of Podocalyxin in TGF-Beta Induced Epithelial Mesenchymal Transition 
PLoS ONE  2011;6(4):e18715.
Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-β induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeletion assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype.
PMCID: PMC3075272  PMID: 21533279
17.  Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells ▿ †  
Journal of Virology  2010;84(20):10888-10906.
Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular “transcriptome.” Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules. We have used stable isotope labeling by amino acids in cell culture (SILAC), combined with high-throughput two-dimensional (2-D) high-performance liquid chromatography (HPLC)/mass spectrometry, to determine quantitative differences in host proteins after infection of human lung A549 cells with human influenza virus A/PR/8/34 (H1N1) for 24 h. Of the 4,689 identified and measured cytosolic protein pairs, 127 were significantly upregulated at >95% confidence, 153 were significantly downregulated at >95% confidence, and a total of 87 proteins were upregulated or downregulated more than 5-fold at >99% confidence. Gene ontology and pathway analyses indicated differentially regulated proteins and included those involved in host cell immunity and antigen presentation, cell adhesion, metabolism, protein function, signal transduction, and transcription pathways.
PMCID: PMC2950599  PMID: 20702633
18.  Investigating the Role of P311 in the Hypertrophic Scar 
PLoS ONE  2010;5(4):e9995.
The mechanisms of hypertrophic scar formation are not fully understood. We previously screened the differentially expressed genes of human hypertrophic scar tissue and identified P311 gene as upregulated. As the activities of P311 in human fibroblast function are unknown, we examined the distribution of it and the effects of forced expression or silencing of expression of P311. P311 expression was detected in fibroblast-like cells from the hypertrophic scar of burn injury patients but not in peripheral blood mononuclear cells, bone marrow mesenchymal stem cells, epidermal cells or normal skin dermal cells. Transfection of fibroblasts with P311 gene stimulated the expression of alpha-smooth muscle actin (α-SMA), TGF-β1 and α1(I) collagen (COL1A1), and enhanced the contraction of fibroblast populated collagen lattices (FPCL). In contrast, interference of fibroblast P311 gene expression decreased the TGF-β1 mRNA expression and reduced the contraction of fibroblasts in FPCL. These results suggest that P311 may be involved in the pathogenesis of hypertrophic scar via induction of a myofibroblastic phenotype and of functions such as TGF-β1 expression. P311 could be a novel target for the control of hypertrophic scar development.
PMCID: PMC2852399  PMID: 20404911
19.  Book Review 
Medical History  2010;54(2):xxi-273.
PMCID: PMC2844282
20.  Rab5 Mediates Caspase-8–promoted Cell Motility and Metastasis 
Molecular Biology of the Cell  2010;21(2):369-376.
Integrins signaling promotes nonapoptotic functions of caspase-8 via activation of small GTPases from the Rab and Rac families. Integrin ligation promotes Rab5 activity, which mediates subsequent activation of Rac1, cytoskeletal remodeling, and enhanced cell motility.
Caspase-8 is a key apical sensory protein that governs cell responses to environmental cues, alternatively promoting apoptosis, proliferation, and cell migration. The proteins responsible for integration of these pathways, however, have remained elusive. Here, we reveal that Rab5 regulates caspase-8–dependent signaling from integrins. Integrin ligation leads to Rab5 activation, association with integrins, and activation of Rac, in a caspase-8–dependent manner. Rab5 activation promotes colocalization and coprecipitation of integrins with caspase-8, concomitant with Rab5 recruitment to integrin-rich regions such as focal adhesions and membrane ruffles. Moreover, caspase-8 expression promotes Rab5-mediated internalization and the recycling of β1 integrins, increasing cell migration independently of caspase catalytic activity. Conversely, Rab5 knockdown prevented caspase-8–mediated integrin signaling for Rac activation, cell migration, and apoptotic signaling, respectively. Similarly, Rab5 was critical for caspase-8–driven cell migration in vivo, because knockdown of Rab5 compromised the ability of caspase-8 to promote metastasis under nonapoptotic conditions. These studies identify Rab5 as a key integrator of caspase-8–mediated signal transduction downstream of integrins, regulating cell survival and migration in vivo and in vitro.
PMCID: PMC2808229  PMID: 19923319
21.  The effects of infliximab therapy on the serum proteome of rheumatoid arthritis patients 
Although the clinical effects of infliximab therapy in rheumatoid arthritis have been documented extensively, the biological effects of this intervention continue to be defined. We sought to examine the impact of infliximab therapy on the serum proteome of rheumatoid arthritis patients by means of a mass spectrometry-based approach.
Sera from 10 patients with rheumatoid arthritis were obtained prior to and following 12 weeks of infliximab therapy using a standard clinical protocol. The sera were immunodepleted of the 12 highest abundance proteins, labeled by the iTRAQ (isobaric tagging for relative and absolute protein quantification) technique, and analyzed by mass spectrometry to identify proteomic changes associated with treatment.
An average of 373 distinct proteins were identified per patient with greater than 95% confidence. In the 3 patients demonstrating the most robust clinical responses, changes of greater than 20% in the serum levels were observed in 39 proteins following treatment. The majority of these proteins were regulated directly or indirectly by tumour necrosis factor-alpha (TNF-α) and nuclear factor-kappa-B, with acute-phase proteins being uniformly down-regulated. A number of proteins, including members of the SERPIN family and S100A8, were down-regulated irrespective of clinical response.
The present study demonstrates that a robust clinical response to infliximab is associated with the down-regulation of a spectrum of serum proteins regulated by TNF-α, and provides a possible basis for defining the broader biological effects of the treatment in vivo.
PMCID: PMC2688177  PMID: 19265537
22.  The identification and characterization of a novel protein, c19orf10, in the synovium 
Joint inflammation and destruction have been linked to the deregulation of the highly synthetic fibroblast-like synoviocytes (FLSs), and much of our current understanding of the mechanisms that underlie synovitis has been collected from studies of FLSs. During a proteomic analysis of FLS cells, we identified a novel protein, c19orf10 (chromosome 19 open reading frame 10), that was produced in significant amounts by these cells. The present study provides a partial characterization of c19orf10 in FLSs, synovial fluid, and the synovium. Murine monoclonal and chicken polyclonal antibodies were produced against recombinant human c19orf10 protein and used to examine the distribution of c19orf10 in cultured FLSs and in synovial tissue sections from patients with rheumatoid arthritis or osteoarthritis. The intracellular staining pattern of c19orf10 is consistent with localization in the endoplasmic reticulum/Golgi distribution. Sections of rheumatoid arthritis and osteoarthritis synovia expressed similar patterns of c19orf10 distribution with perivascular and synovial lining staining. Double-staining in situ analysis suggests that fibroblast-like synovial cells produced c19orf10, whereas macrophages, B cells, or T cells produced little or none of this protein. There is evidence of secretion into the vascular space and the extracellular matrix surrounding the synovial lining. A competitive enzyme-linked immunosorbent assay confirmed the presence of microgram levels of c19orf10 in the synovial fluids of patients with one of various arthropathies. Collectively, these results suggest that c19orf10 is an FLS-derived protein that is secreted into the synovial fluid. However, the significance of this protein in synovial biology remains to be determined. The absence of known structural motifs or domains and its relatively late evolutionary appearance raise interesting questions about its function.
PMCID: PMC1906808  PMID: 17362502
23.  Anti-Sa antibodies: prognostic and pathogenetic significance to rheumatoid arthritis 
Arthritis Research & Therapy  2004;6(2):86-89.
Anti-Sa antibodies are detected in the serum of 20–47% of patients with rheumatoid arthritis. These antibodies have a high degree of specificity for the disease, and appear to identify a subset of early rheumatoid arthritis patients destined to have aggressive and destructive disease. It has recently been confirmed that anti-Sa antibodies are directed to citrullinated vimentin, thus placing them in the anti-citrulline family of autoantibodies. The Sa antigen has previously been shown to be present in synovium. This, along with the demonstration of citrullinated proteins in rheumatoid synovium, suggests that anti-Sa antibodies may play a pathogenetic role in the initiation and/or persistence of rheumatoid synovitis.
PMCID: PMC400444  PMID: 15059270
anti-citrulline antibodies; anti-Sa; autoantibodies; prognosis; rheumatoid arthritis; synovium
24.  The synovial proteome: analysis of fibroblast-like synoviocytes 
Arthritis Research & Therapy  2004;6(2):R161-R168.
The present studies were initiated to determine the protein expression patterns of fibroblast-like synovial (FLS) cells derived from the synovia of rheumatoid arthritis patients. The cellular proteins were separated by two-dimensional polyacrylamide gel electrophoresis and the in-gel digested proteins were analyzed by matrix-assisted laser desorption ionization mass spectrometry. A total of 368 spots were examined and 254 identifications were made. The studies identified a number of proteins that have been implicated in the normal or pathological FLS function (e.g. uridine diphosphoglucose dehydrogenase, galectin 1 and galectin 3) or that have been characterized as potential autoantigens in rheumatoid arthritis (e.g. BiP, colligin, HC gp-39). A novel uncharacterized protein product of chromosome 19 open reading frame 10 was also detected as an apparently major component of FLS cells. These results demonstrate the utility of high-content proteomic approaches in the analysis of FLS composition.
PMCID: PMC400437  PMID: 15059280
autoantigens; fibroblast-like synovial cells; galectins; matrix-assisted laser desorption ionization mass spectrometry; proteomics
25.  A Role for Caveolin and the Urokinase Receptor in Integrin-mediated Adhesion and Signaling  
The Journal of Cell Biology  1999;144(6):1285-1294.
The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with β1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with β1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes β1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, β1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with β1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal β1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.
PMCID: PMC2150580  PMID: 10087270
urokinase receptor; caveolin; integrins; adhesion; cell signaling

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